Much remains to be learned about the events responsible for the survival, expansion and differentiation of CD25plusCD4minusCD8minusthymocytes. A signal delivered by the pre-TCR is known to be sufficient, but not necessary, to reach the CD25minus CD4plus CD8plus stage in vivo. Recent observations suggest that CD81 expressed by thymic epithelial cells also plays a pivotal role in this process. It has been clearly shown that maturation of murine CD25plusCD4minusCD8minus cells to CD25minusCD4plusCD8plus cells can be induced in reaggregation culture by a CD81plus fetal thymic epithelial cell line as well as by other cell types transfected with a murine CD81 cDNA. These findings established that CD81 can transmit a developmental signal to CD4minusCD8minus cells possibly through a putative receptor. A combination of molecular and biochemical approaches will be employed for the identification of potential CD81 receptors expressed by CD4minusCD8minus thymocytes. The developmental relationship between pre-TCR and CD81 mediated signals will be investigated by evaluating the status of TCR-beta gene rearrangement in thymocytes recovered from anti-CD81 antibody treated fetal thymic organ cultures. These experiments should help provide a model to explain the demonstrated requirement of pre-TCR and CD81-mediated signals in early T cell development. Finally, the function of CD81 in early T cell development will be analyzed in vivo following antibody treatment and by using CD81 deficient mutant mice generated using the Cre-loxP system.